Bovine leukaemia virus (BLV) belongs to the
It is known that infectious BLV particles and/or somatic cells infected with BLV can be transmitted to calves by milk, but it is thought to be a minor route of infection. BLV infection affects production efficiency and causes decrements of immunological status, and the sanitary and economic impact of infection is associated with hindered international movement of cattle. In many European countries, national programs controlling the spread of BLV infection and eradicating the disease exist, and because of these recommendations the cattle population is relatively free from BLV (25). In May 2017, Poland was recognised as free from BLV infection.
Flow cytometry (FC) is a technique for qualitative and quantitative assessment of multiple parameters of individual cells or particles in different complex cell suspensions. The technology is an integral component of research in the life sciences, particularly immunology, and is in common use for diagnostic and prognostic investigations in human oncology (specifically in haematooncological diagnosis). Recently, the flow cytometry technique has been integrated into veterinary oncology and immunology. Flow cytometry determination of BLV glycoprotein (gp) 51 expression in blood and milk lymphocytes of BLV-infected cows was the aim of the study.
All cows were serologically positive and the results of molecular investigations showed the presence of proviral DNA. On the basis of haematological analysis, the animals fell into two groups: in eleven cows the lymphocyte levels were in the range of 7,600– 11,200 cells per mm3 (mean value 9,354 cells per mm3) and these cows were classified as AL, and in the other eight cows very high lymphocyte values were determined in the range of 12,800–63,200 cells per mm3 (mean value 29,650/mm3) and the animals were classified as PL.
The results of flow cytometry determination of peripheral blood subpopulations are presented in Table 1.
The percentage of peripheral blood lymphocyte subpopulations of bovine leukaemia virus–infected cattle in the aleukaemic (AL) and persistently lymphocytotic (PL) stages
Cow sample | ELISA | PCR | Phase of infection | Lymphocytes/mm3 | CD19+ | CD19+ IgM+ | CD4+ | CD8+ |
---|---|---|---|---|---|---|---|---|
1 | + | + | AL | 7,600 | 25 | 46 | 25 | 3 |
2 | + | + | 7,800 | 67 | 75 | 15 | 17 | |
3 | + | + | 8,400 | 54 | 80 | 27 | 23 | |
4 | + | + | 8,800 | 45 | 27 | 31 | 10 | |
5 | + | + | 9,000 | 54 | 45 | 26 | 32 | |
6 | + | + | 9,600 | 54 | 61 | 23 | 27 | |
7 | + | + | 9,600 | 67 | 64 | 33 | 20 | |
8 | + | + | 10,200 | 47 | 18 | 30 | 6 | |
9 | + | + | 10,300 | 82 | 68 | 1 | 2 | |
10 | + | + | 10,400 | 96 | 20 | 2 | 2 | |
11 | + | + | 11,200 | 51 | 19 | 30 | 4 | |
Mean value | 9,354 | 58.3 | 47.5 | 22.1 | 13.5 | |||
1 | + | + | PL | 12,800 | 66 | 65 | 21 | 4 |
2 | + | + | 15,200 | 55 | 77 | 27 | 31 | |
3 | + | + | 15,600 | 39 | 62 | 13 | 5 | |
4 | + | + | 26,000 | 83 | 81 | 6 | 2 | |
5 | + | + | 26,400 | 61 | 81 | 5 | 2 | |
6 | + | + | 39,000 | 96 | 61 | 8 | 2 | |
7 | + | + | 39,000 | 73 | 36 | 7 | 2 | |
8 | + | + | 63,200 | 96 | 86 | 4 | 4 | |
Mean value | 29,650 | 71.1 | 68.6 | 11.4 | 6.5 |
CD – cluster of differentiation; IgM – immunoglobulin M
On the basis of flow cytometry analysis, it was found that the expression of BLV gp51 in infected cattle was mainly in CD19+ and CD19+ IgM+ lymphocytes. In the AL stage of the disease, a lower percentage of CD19+ (58.3%) and CD19+ IgM+ (47.5%) B lymphocytes was found than in the PL stage (71.1% and 68.6% respective mean values).
The opposite dependency was demonstrated for CD4+ and CD8+ T lymphocytes: their higher percentages (22.1% and 13.5%, respectively) were found in the AL stage of infection and their lower ones (11.4% and 6.5%, respectively) in the PL stage (Fig. 1). A detailed analysis of the infection stage effect on the differentiation of the subpopulation sizes of PBLs revealed a significantly lower number (P = 0.005 and P = 0.002) of CD19+ and CD19+ IgM+ B lymphocytes as well as a negligibly lower number (not statistically confirmed, with P = 0.348 and P = 0.743) of CD4+ and CD8+ T lymphocytes in animals in the aleukaemic stage, compared to animals with persistent lymphocytosis.
The difference in the percentages of cluster of differentiation CD19+ and CD19+ immunoglobulin M+ B and CD4+ and CD8+ T lymphocyte subpopulations depending on bovine leukaemia virus infection stage
AL – aleukaemic stage of infection; PL – persistently lymphocytotic stage of infection. Median values were used to provide a better measure of the central tendency
The ratio of CD4+ to CD8+ T lymphocytes was lower in aleukaemic animals (2.80) than in persistently lymphocytotic animals (2.84). However, the difference between these values was not confirmed statistically (P = 0.432). The ratio of CD19+ to CD19+ IgM+ B lymphocytes was not significantly different for cows in the AL stage (1.66) from this ratio in animals in the PL stage (1.11) (P = 0.363). Thus, an increase in the number of CD19+ IgM+ cells coincided with an increase in total B lymphocytes (Tables 1 and 2).
The expression of gp51 in blood lymphocyte subsets of bovine leukaemia virus infected cattle (%)
Cow sample | Stage of infection | Lymphocytes/mm3 | CD markers + gp51 (%) |
Cells with gp51 expression | |||
---|---|---|---|---|---|---|---|
CD19+ | CD19+ IgM+ | CD4+ | CD8+ | ||||
1 | AL | 7,600 | 15 | 20 | 0 | 16 | 51 |
2 | 7,800 | 25 | 7 | 1 | 7 | 40 | |
3 | 8,400 | 29 | 20 | 0 | 10 | 59 | |
4 | 8,800 | 38 | 12 | 2 | 10 | 62 | |
5 | 9,000 | 30 | 18 | 3 | 21 | 72 | |
6 | 9,600 | 38 | 13 | 0 | 17 | 68 | |
7 | 9,600 | 1 | 3 | 0 | 2 | 6 | |
8 | 10,200 | 24 | 10 | 0 | 1 | 35 | |
9 | 10,300 | 3 | 3 | 0 | 0 | 6 | |
10 | 10,400 | 28 | 19 | 3 | 12 | 62 | |
11 | 11,200 | 1 | 1 | 0 | 0 | 2 | |
Mean value | 9,354 | 21.0 | 11.4 | 0.8 | 8.7 | 41.3 | |
1 | PL | 12,800 | 16 | 10 | 1 | 4 | 31 |
2 | 15,200 | 21 | 24 | 0 | 17 | 62 | |
3 | 15,600 | 19 | 30 | 0 | 5 | 54 | |
4 | 26,000 | 31 | 9 | 0 | 2 | 42 | |
5 | 26,400 | 9 | 8 | 1 | 5 | 23 | |
6 | 39,000 | 34 | 23 | 2 | 2 | 61 | |
7 | 39,000 | 28 | 18 | 0 | 2 | 58 | |
8 | 63,200 | 68 | 20 | 1 | 4 | 93 | |
Mean value | 29,650 | 28.25 | 17.7 | 0.6 | 5.2 | 53.0 |
CD – cluster of differentiation; IgM – immunoglobulin M; AL – aleukaemic; PL – persistently lymphocytotic
Additionally, the B cell to T cell ratios were lower in aleukaemic cattle (10.5) than in animals in the PL stage (16.4). These differences between ratios were caused by an excessive proliferation of immature CD19+ IgM+ cells and a fall in the CD8+ T lymphocyte percentage (Fig. 1).
In the PL stage of infection, strong proliferation and accumulation of B lymphocytes and expression of both CD19+ and CD19+ IgM+ markers were detected. The association of these two markers as well as CD4+ and CD8+ markers in PBLs obtained from BLV-infected cows and the expression of BLV gp51 antigen in these cells were examined in two-colour flow cytometry and the results are presented in Table 2. The percentage of CD19+ B cells which expressed BLV gp51+ antigen depended on the progress of BLV infection and increased from 21.0% in the AL to 28.25% in the PL stage. Similarly, the percentage of CD19+ IgM+ B cells with BLV gp51+ antigen expression increased from 11.4% in the AL to 17.7% in the PL stage of infection (Table 2). An opposite tendency was observed with BLV gp51+ antigen expression in CD4+ and CD8+ T cells, the higher percentages of which (0.8% and 8.7%, respectively) were noted in the AL stage of infection and the lower values (0.6% and 5.2%, respectively) in the PL stage (Figs 2, 3A and 3B).
The effect of BLV gp51 antigen expression in the of cluster of differentiation CD19+ and CD19+ immunoglobulin M (IgM)+ B and CD4+ and CD8+ T lymphocyte subpopulations
AL – aleukaemic stage of infection; PL – persistently lymphocytotic stage of infection
The effect of BLV gp51 antigen expression in the number of cluster of differentiation CD19+ and CD19+ immunoglobulin M (IgM)+ B and CD4+ and CD8+ T lymphocyte subpopulations
AL – aleukaemic stage of infection; PL – persistently lymphocytotic stage of infection
An analysis of the presence of the gp51 antigen in blood lymphocytes revealed a significantly lower number of CD4+ T cells expressing it in the aleukaemic stage (76/mm3, 0.8%) (P < 0.003 and P < 0.004, respectively) compared to the numbers of CD19+, CD19+ IgM+ and CD8+ cells expressing the gp51 antigen in this stage (1,918/mm3, 21.0%; 1,034/mm3, 11.4%; and 777/mm3, 8.7% respectively). The analysis also discovered a significantly lower number of CD4+ gp51+ cells in the PL stage of infection (226/mm3, 0.6%) (P < 0.008 and P < 0.03, respectively) in comparison with the numbers of B and CD8+ cells expressing this antigen in this stage (10,726/mm3, 28.25%; 5,336/mm3, 17.7%; and 1,226/mm3, 5.2% respectively) (Fig. 3A and B). It was observed that the weakening of the expression of CD4+ gp51+ lymphocytes in peripheral blood correlated with the fall in the CD4+ gp51+ to CD8+ gp51+ ratio. Long-term infections showed a stronger contraction of the CD4+ gp51+ and CD8+ gp51+ lymphocyte subsets that also included a decrease in the absolute size of the CD4+ cell population and the absolute number of CD8+ cells. The results concerning the expression of the gp51 antigen in cells from milk are presented in Table 3 and a comparison of this expression in blood and milk is illustrated in Fig. 4. The expression of gp51 in CD19+ and CD19+ IgM+ cells from milk in the AL stage was low: the respective mean values were 3.5% and 1.8%. In animals remaining in the PL stage, these values increased and the maximal percentages found in persistent lymphocytosis for CD19+ and CD19+ IgM+ were 6.3% and 2.7%, respectively.
The expression of glycoprotein 51 in blood and milk lymphocytes of bovine leukaemia virus-infected cows with clearly much higher expression in blood lymphocytes than in milk cells
The expression of gp51 in milk lymphocyte subsets of bovine leukaemia virus–infected cows (%)
Cow sample | Stage of infection | Lymphocytes/mm3 | CD markers (%) |
Expression of gp51 in milk lymphocytes (%) | |||||
---|---|---|---|---|---|---|---|---|---|
ELISA | PCR | CD19+ | CD4+ | CD8+ | CD19+ IgM+ | ||||
1 | AL | 7,600 | + | + | 1 | − | − | 1 | 2 |
2 | 7,800 | + | + | 2 | − | − | − | 2 | |
3 | 8,400 | + | + | − | − | − | 3 | 3 | |
4 | 8,800 | + | + | − | − | − | 2 | 2 | |
5 | 9,000 | + | + | 2 | − | 1 | − | 3 | |
6 | 9,600 | + | + | 1 | − | 1 | − | 2 | |
7 | 9,600 | + | + | 1 | − | − | 3 | 4 | |
8 | 10,200 | + | + | 1 | − | 1 | 1 | 3 | |
9 | 10,300 | + | + | 3 | − | − | − | 3 | |
10 | 10,400 | + | + | 18 | − | − | 4 | 21 | |
11 | 11,200 | + | + | 10 | − | − | 6 | 20 | |
Mean value | 9,354 | + | + | 3.5 | - | 0.3 | 1.8 | 5.7 | |
1 | PL | 12,800 | + | + | 1 | − | − | 1 | 2 |
2 | 15,200 | + | + | 3 | − | − | 2 | 5 | |
3 | 15,600 | + | + | 1 | − | − | 2 | 3 | |
4 | 26,000 | + | + | 2 | − | 1 | − | 3 | |
5 | 26,400 | + | + | 9 | 1 | 2 | 3 | 15 | |
6 | 39,000 | + | + | 4 | − | − | 3 | 7 | |
7 | 39,000 | + | + | 10 | − | 1 | 2 | 13 | |
8 | 63,200 | + | + | 21 | − | 4 | 9 | 33 | |
Mean value | 29,650 | 6.3 | 0.1 | 1.0 | 2.7 | 10.2 |
CD – cluster of differentiation; IgM – immunoglobulin M; AL – aleukaemic; PL – persistently lymphocytotic
In animals in the AL stage, gp51 expression was determined in a mean 41.3% of blood lymphocytes. The highest mean values of gp51 expression were determined in CD19+ lymphocytes at 21.0% and CD19+ IgM+ lymphocytes at 11.4%. Cells of CD4+ type expressed the antigen in a mean 0.8% of their population and CD8+ lymphocytes did so in 8.7% of cells (also the mean value).
The presence of gp51 in milk lymphocytes of animals in the AL stage was detected in 2%–21% of cells, the mean value being 5.7%. This glycoprotein was present in 3.5% of cells with CD19+ antigen and in 1.8% of CD19+ IgM+ lymphocytes. Glycoprotein 51 was absent from CD4+ lymphocytes, and only in low presence in CD8+ lymphocytes, where 0.3% of cells had this protein detectable. In milk samples of cows in the PL stage of leukaemia, the presence of gp51 in milk was detected in 2%–33% of milk lymphocytes for a mean value of 10.2%. The expression of gp51 was detected in 6.2% of CD19+ and in 2.7% of CD19+ IgM+ lymphocytes. In CD4+ T cells, this expression was found in 0.1% and in CD8+ T lymphocytes in 1.0% (Fig. 5A and 5B).
The expression of BLV glycoprotein (gp) 51 antigen in milk: in cluster of differentiation CD19+, CD19+ immunoglobulin M (IgM)+ B and CD4+, CD8+ T lymphocyte subpopulations
A – AL stage of infection; B – PL stage of infection
The highest gp51 expression was determined in milk lymphocytes of cows with high-count lymphocytosis: there were 21% of CD19+, 9% of CD19+ IgM+ and 4% of CD8+ cells. The results indicated that CD19+ and CD19+ IgM+ lymphocytes are the main target cells, because this was where BLV gp51 expression was predominantly detected. These results are presented in Table 3 and Fig. 4. The presence of green fluorescence in smears stained with conjugate attached to anti-gp51 monoclonal antibodies confirmed the presence of BLV gp51 on blood and milk lymphocytes (Figs 6 and 7).
Immunofluorescence showing the expression of bovine leukaemia virus glycoprotein 51 in blood lymphocytes of a cow with persistent lymphocytosis
Immunofluorescence showing the expression of bovine leukaemia virus glycoprotein51 in cells from a milk sample from a leukaemic cow
The results of our investigations indicated that the expression of BLV gp51 was present in blood and milk lymphocytes of animals infected with BLV. The gp51 expression was much higher in lymphocytes from blood than in those from milk. The studies performed by Bartlett
Our results showed that gp51 expression in blood lymphocytes of cows in the AL stage of bovine leukaemia was detected mainly in CD19+ and CD19+ IgM+ lymphocytes, which indicated BLV tropism for B cells and underlined the role of B lymphocytes in BLV infection. The gp51 antigen was also detected in T cells, but the expression was not strong, which confirmed that T cells can also be the source of viral particles. Meirom
Bovine leukaemia virus induces abnormal B cell proliferation and B cell lymphoma in cattle, where the BLV provirus is integrated with the genome of the host. BLV-infected B lymphocytes rarely express viral proteins
The expression of gp51 in blood and milk lymphocytes of cows with persistent lymphocytosis was much stronger than in aleukaemic animals. This expression was visible predominantly in CD19+ and CD19+ IgM+ lymphocytes. The obtained results indicated the role of milk in BLV transmission in the herds. Although BLV demonstrates tropism for B cells, it can affect both adaptive and innate immunities because these systems share many effector mechanisms. Mammary gland immunity is largely dependent upon neutrophilic functions. Many studies have investigated the effects of BLV infection on lymphocyte subsets in infected animals (9, 29, 30), B cell vitality (11, 28, 31) and neutrophil functions (10, 28). Bovine leukaemia virus can be the reason for changes in milk production and fertility in cows. Many authors indicated its negative economic impact on cattle farming. Mastitis and decreased milk production have been associated with BLV infection, particularly in BLV-infected animals with persistent lymphocytosis and on high-performing infected dairy farms (11, 35). Infection with BLV caused economic losses not only in its effect on milk production but also in impairing animal reproduction, which was indicated by many researchers (33, 35); however, in previous investigations other authors found no differences in milk production by BLV+ and by BLV− cows.
Investigations in Canada by Nekouei
Infection with BLV also has been shown to alter the expression of other cytokines besides IL-4, IL-6 and IL-10, namely IL-2 and IL-12. The virus’ regulation of several immunomodulators provides multiple mechanisms for it to suppress immunity in affected cattle (14). Interleukin 6 may play a contributory role in BLV latency due to its elevation in blood (14, 30). Additionally, IL-6 mediates hypoferraemia developing in inflammation through reduction of hepcidin, which reduces intestinal iron absorption and releases iron from macrophages (24). High ferritin concentrations were detected in sera of BLV-infected cows (26). Ferritin can be a tumour marker in humans and a useful factor to monitor treatment effectiveness (1).
Many authors investigated the relationship between BLV and tumours in humans and the possibility of human infection with BLV after milk and meat consumption or in association with work on cattle farms or abattoirs. Maruyama
Bovine leukaemia virus capsid proteins were detected in human serum by Buehring
On the other hand, other investigations by PCR did not find BLV in breast cancer in China. With the use of whole-genome sequencing of DNA from 51 breast cancer tissue samples, Gillet
The results of investigations performed by Yamada
There are many active molecules such as growth factors and cytokines present in bovine milk and as many as 2,107 proteins in bovine milk exosomes have been identified. It is evident that bovine milk exosomes play important roles in the growth of infants, such as driving immune system maturation and responsiveness. Zhou
Bovine milk exosomes may be useful and serve as biomarkers of the physiological state and infectious diseases. Yamada
Infection with BLV is correlated with the inhibition of apoptosis, and leads to the generation of a reservoir of infected cells. This circumstance and BLV tropism for B lymphocytes determined in infected animals may explain the lower number of milk B cells, especially in the PL stage, which were undergoing apoptosis. This phenomenon was observed in blood B cells by many authors (11, 14). These studies indicate the importance of monitoring and eradication of BLV infections in cattle, especially for human health and the minimisation of economic losses.